The long-term goals of our research are to delineate biochemical steps in key early events during HIV replication. To this end, we will continue our efforts on dissecting the structural determinants of critical functions performed by viral reverse transcriptase (RT) and we will expand our original observation of RT interaction with the viral integrase (IN) to understand its virological significance. Three specific aims are proposed. Aim (1) RT determinants of dNTP selection: We have made a new observation that the fingers beta3-Deta4 hairpin loop, specifically the contact between K65 residue and the gamma phosphate of incoming dNTP, is responsible for RT's low fidelity. We will use pre-steady state kinetics to generate evidence that the insertion fidelity is governed at the level of initial recognition of correct vs. incorrect dNTPs. Forward mutation rates and mispair extension efficiencies will be measured to determine if the increased fidelity extends beyond dNTP misinsertion to other forms of errors. We will also employ halogenated analogs of dNTPs to determine the role of active site tightness in dNTP selection. These studies should enhance our knowledge of the structural determinants of the RT's active site. Aim (2) Determinants of strand displacement DNA synthesis: We hypothesize that F61 and W24 residues control strand displacement synthesis by 'gating' the duplex ahead of the active site. Mutant RTs with natural and unnatural amino acid substitutions (at F61 or W24) with variable predicted abilities to stabilize the terminal base pair will be used to examine their strand displacement synthesis in vitro. In virological experiments, effect of mutations that affect strand displacement during viral replication will be studied. Aim (3) Functional role of RT-IN Interaction in HIV replication: Using reverse yeast 2-hybrid screen, we will isolate interaction-defective mutants of RT or IN. Mutations will be built into viral clones to examine the importance of RT-IN interaction in HIV replication.